Figure 7.

Replacement of the At GID1b Loop with the At GID1a Loop Abolishes GA-Independent GID1–GAI Interaction.

(A) Y2H assay using mutated At GID1s as bait and GAI as prey in the presence or absence of GA₄. In At GID1b (1a-loop), the entire loop region of GID1b (from S84 to T103) was replaced with the corresponding region from At GID1a. In At GID1b^R90P, At GID1b^{R90P and H91H}, and At GDI1b^H91P, amino acids Arg-90 and/or His-91 were replaced with the corresponding residues from At GID1a, as indicated. β-Gal activity was determined by a liquid assay with yeast strain Y187 transformants (means ± sd; n = 3). Equal expression level of bait proteins in yeast cells was confirmed by immunoblot analysis (see Supplemental Figure 6B online).

(B) Various kinetic values; k_a (association rate), k_d (dissociation rate), and K_D (k_d/k_a, dissociation constant), of At GID1a– and At GID1b–GA₄ interactions by SPR.